Mitosis is a basic biological process with considerable biomedical importance, yet its molecular mechanisms are still poorly understood. Spindle structure ha been thoroughly described, but spindle biochemistry is still poorly defined for want for good assays to determine the importance of putative spindle components. We propose to seek new spindle proteins using monoclonal antibodies prepared against specific components of several complex antigens: microtubule associated proteins, extracts of sea urchin spindles and flagella, and eluants from affinity columns. Positive clones will be further screened by immunofluorescense to test for production of antibodies that strain the spindle of mitotic cells. Clones positive by this assay will receive careful immunochemical analysis by electrophoretic gel blotting methods, immunoprecipitation, and adsorption chromatography to characterize their reactivity with mammalian cell extracts and to identify potential spindle proteins. Such proteins or their antibodies will be isolated by affinity chromatogrphy, labeled with fluorescent dyes and injected by microneedles into mitotic PtK cells to follow their incorporation into the spindle. We will use quantitative optical analysis obtained from video processing of light microscope images to measure changes in spindle structure and behavior as seen with fluorescense, differential interference contrast, and polarization optics. Thus we can look for functional effects of our microinjections upon normal spindle behavior and gain insight into the importance for mitosis of each component we identify. Further tests for component function will be sought by injecting the proteins or their antibodies into spreading cultured cells or by applying them to lysed cell models, such as flagella or myofibrils. Relationships between multiple components will be studied through their reactions in vitro and their relative positions in vivo as seen by double label immunoelectron microscopy, crosslinking studies, and fluorescence energy transfer. In these ways we hope to identify new spindle proteins and characterize their functions in mitosis, thereby improving the current knowledge of spindle biochemistry and the cellular mechanisms for chromosome movement.